Project Summary This K08 Career Development Award application is intended to support the acquisition of skills and knowledge needed to fulfill my long-term goal of becoming an independent physician-scientist focused on combating antimicrobial resistant organisms, a serious threat to medical practice worldwide. Vancomycin resistant enterococci (VRE) are an example of these pathogens and are a leading cause of healthcare associated infections affecting critically ill and immunocompromised patients. VRE are categorized by the CDC as a serious threat requiring the urgent development of novel therapeutic strategies. The lipopeptide antibiotic daptomycin (DAP) is now a front line agent for VRE infections, but resistance to DAP can arise while on therapy. The LiaFSR system, a major mediator of the cell envelope stress response, has been strongly implicated in the development of DAP resistance. Inactivation of this system by deletion of the gene encoding the LiaR response regulator was shown to re-sensitize enterococci to DAP. However, adaptation of LiaR deficient strains of both clinical and laboratory origin resulted in DAP resistance, suggesting that alternate pathways can protect the cell from antibiotic attack. Using whole genome sequencing of adapted strain pairs, I identified two pathways with novel contributions to DAP and cephalosporin resistance in enterococci, i) the YxdJK stress response system, and ii) the dihydroxyacetone kinase (DAK) domain protein involved in the metabolism of extracellular fatty acids. The YxdJK system consists of a sensor histidine kinase (YxdK), a DNA binding response regulator (YxdJ) and two ATP-binding cassette (ABC) transporters required to confer resistance to bacitracin. Deletion of the gene encoding the YxdJ response regulator sensitizes Enterococcus faecalis to both DAP and cephalosporins, despite a functional LiaFSR system. DAP-resistant strains using the above pathways appear to display a very distinct mechanism of resistance to cell-envelope acting antibiotics. This proposal is designed to dissect the role of the YxdJK system and the DAK enzyme in two major specific aims. First, I will characterize the contributions of the YxdJK system to the cell envelope stress response to antibiotics by defining how the system senses antibiotic stress and what genes are differentially expressed when the system is active. Second, I will determine the DAK mediated changes that alter membrane susceptibility to antibiotics, by comparing the membranes of wild type and DAK deletion strains to assess for changes in phospholipids, envelope structure, membrane protein function, and biofilm formation. The Center for Antimicrobial Resistance and Microbial Genomics (CARMiG) at the University of Texas Health Science Center and adjacent institutions of the Texas Medical Center will provide an unparalleled environment to grow as an investigator, with both an institutional commitment to combating antimicrobial resistance and an intensive mentorship program dedicated to helping junior faculty make the transition to independence.